S-nitrosylation of proteins in hippocampal and cortical neurons in excitotoxicity: differential regulation of NF-kB

Abstract

Cell death by excitotoxicity is a consequence of excessive stimulation of N-methyl-Daspartate (NMDA) receptors, causing calcium overload and synthesis of nitric oxide. Hippocampal neurons are selectively sensitive to excitoxicity while other neuronal types, including cortical cells, are resistant. Nitric oxide can regulate protein function by by thiol nitrosylation, targeting mainly cysteine residues in proteins. We have thus focused on the identification of S-nitrosylated proteins in cortical and hippocampal neuronal cultures by mass spectrometry of proteins pulled down by the biotin switch method. In total, 178 and 360 proteins were identified in hippocampal and cortical cultures, respectively in any experimental condition. From these proteins, 93 were common to both culture types. To identify possible NMDA-regulated proteins and signaling pathways, the GeneCodis and Gene Ontology software was used. Interestingly, the meta-analysis strongly suggested that S-nitrosylation regulates different biological processes, and possibly transcriptional activity mediated by NF-κB. Selected proteins were quantified by Western blot analysis. We found that the S-nitrosylation level of the synaptic scaffolding protein SAPAP-4 (discs, large (Drosophila) homolog-associated protein 4 [DLGAP4_RAT]) increased after NMDA exposure in cortical, but not in hippocampal neurons. In turn, an opposing regulation occurred in the p65 subunit of NF-κB in both culture types: its Snitrosylation increased in cortical neurons exposed to NMDA, thus suggesting inactivation of transcriptional activity, but decreased in hippocampal neurons, thus suggesting augmented transcriptional activity. This was confirmed when the nuclear translocation of p65 was studied by cellular fractionation, immunocytochemistry and quantification by qRT-PCR of the NF-kB downstream genes Bax and Caspase 11. Moreover, the endothelial isoform of nitric oxide synthase, that is expressed at higher levels in cortical neurons, contributes significantly to protein S-nitrosylation. Our proteomic analysis indicates that protein Snitrosylation is a regulated and ubiquitous post-translational modification while the differential S-nitrosylation of NF-κB may contribute importantly to the differential susceptibility of hippocampal and cortical neurons to excitotoxicity. Key words: excitotoxicity, S-nitrosylation, proteomics